Nobuhiko Asada

Activation of prophenoloxidase with 2-propanol and other organic compounds in Drosophilamelanogaster

Activation with 2-propanol and other organic compounds of prophenoloxidase purified from pupae of Drosophila melanogaster was analyzed. A1, one of the two isozymes of the prophenoloxidase, could be activated with both an endogenous activating system and artificial organic compounds including alcohols. A1 was activated within 2 min after addition of 2-propanol. The phenoloxidase activity of A1, which had been activated with 2-propanol, decreased gradually by lowering the concentration of 2-propanol taking c 60 min to attain a low level, and the activity could be re-elevated at the re-introduction of 2-propanol. Thus the reversibility of the activation of A1 in response to the change of the concentration of 2-propanol in the activating mixture could be observed. Optimum concentration of 2-propanol for the rate of activation was 50%, optimum temperature was 30 degrees C and optimum pH was 7.5. The final level of the phenoloxidase activity, which had been activated with 2-propanol, was higher than that activated with the endogenous activating system. The activated state of A1 showed properties of a tyrosinase-type phenoloxidase. The results suggested that the activation of A1 with 2-propanol is caused by the reversible conformational change of the prophenoloxidase molecule.

Properties of Phenoloxidases Generated from Prophenoloxidase with 2-Propanol and the Natural Activator in Drosophila melanogaster

Prophenoloxidases A1 andA3 in Drosophila melanogaster were activatedwith 2-propanol and a partially purified naturalactivator. For prophenoloxidase activation, the optimumtemperaturewas 30 degrees C and the optimum pH was 8. Bothmono- and diphenoloxidase activities were found inA1 and A3 activated with2-propanol, whereas only diphenoloxidase activity wasdetected inA3 activated with a naturalactivator. The kinetic properties, Km and Vmax, were not similar in those phenoloxidasesactivated with different activating agents. The rateof inhibition of phenoloxidase bydiethyldithiocarbamate and phenylthiocarbamate dependedon the concentration of 2-propanol. Both compoundsexhibited a noncompetitive pattern ofinhibition.

Prophenol Oxidase A3 in Drosophila melanogaster: Activation and the PCR-Based cDNA Sequence

Phenol oxidase exists in Drosophila hemolymph as a prophenol oxidase, A1 and A3, that is activated in vivo with a native activating system, AMM-1, by limited proteolysis with time. The polypeptide in purified prophenol oxidase A3 has a molecular weight of approximately 77,000 Da. A PCR-based cDNA sequence coding A3 has 2501 bp encoding an open reading frame of 682 amino acid residues. The potential copper-binding sites, from Trp-196 to Tyr-245, and from Asn-366 to Phe-421, are highly homologous to the corresponding sites in other invertebrates. The availability of prophenol oxidase cDNA should be useful in revealing the biochemical differences between A1 and A3 isoforms in Drosophila melanogaster that are refractory or unable to activate prophenol oxidase.

Effect of Ionic Concentration on the Higher-Order Structure of Prophenol Oxidase in Drosophila melanogaster

Phenol oxidase in Drosophila melanogaster occurs as precursors designated prophenol oxidases A1 and A3. Crossing experiments between isozyme variants proved that prophenol oxidase in this species is a homodimer. Prophenol oxidases were partially purified using ammonium sulfate fractionation, phenyl Sepharose, and DEAE-cellulose column chromatography. The preparations were mixed, then dialyzed against buffer containing varying salt concentrations. The resulting prophenol oxidase was analyzed by gel electrophoresis. At 20 mM KCl or NaCl, two bands of phenol oxidase were observed, corresponding to the parental ones as monomer, whereas at 200 mM KCl or NaCl, three bands appeared in the gel, one being a dimer. The monomer–dimer reversibility of the Drosophila prophenol oxidase depends on the salt concentrations. The phenol oxidase activity remained unaffected within the KCl concentrations tested. Considering the ionic concentration of Drosophila hemolymph, these results indicate that prophenol oxidase exists as a dimer in vivo, and the higher-order structure of prophenol oxidase can be altered reversibly by ionic concentrations in vitro.

Deleterious effect of null phenoloxidase mutation on the survival rate in Drosophila melanogaster.

The effect of null activity of phenoloxidase on the survival rate was investigated in mutants of Drosophila melanogaster. MoxGM95 and Dox-3KD95, structural genes for prophenoloxidase A1 and A3, were found in natural populations in the former Soviet Union, and affected the phenoloxidase activity in active A1 or A3, respectively. After linking the visible markers located on the second chromosome together with the variants, cross experiments were performed to make homozygote, rdo Dox-3KD95 pr C MoxGM95 wt. No double mutant had emerged. In the mutant, c MoxGM95 wt Pu2, the viability was greatly reduced. These results suggested that phenoloxidase and tyrosine-3-hydroxylase act as indispensable proteins to maintain life in Drosophila.

Genetic variants affecting phenoloxidase activity in Drosophila melanogaster.

In Drosophila melanogaster, two new variants affecting the activity of phenoloxidase were found in natural populations at Gomel in Belorussia and at Krasnodar in Russia. Prophenoloxidases, A1and A3, in these variants had the same mobilities on native electrophoresis as the wild type. However, enzymatic activities in their activated states were much lower than in the wild type, whereas the existence of prophenoloxidase proteins was demonstrated. Egg-to-adult and relative viabilities in the variants did not decrease at temperatures between 18 and 29°C. Genetic analyses indicated that the genes showing the phenotype of variants are new alleles of Mox and Dox-3 on the second chromosome.

Isoform-dependent phenol oxidase activity during the developmental stages in Drosophila melanogaster.

In many arthropods, the stage that hatches from the egg has unique features that distinguishes it from subsequent life stages. Insect metamorphosis is a fascinating and highly successful biological adaptation, but there is much uncertainty as to how it is regulated. In the fruit fly, Drosophila melanogaster , the developmental timing within the third-instar larva appears to be dependent on enzymatic regulation for pupariation. Phenol oxidase is the enzyme that catalyzes the conversion of tyrosine to dopa, an essential precursor for several dopa and dopamine derivatives necessary for sclerotization of the new cuticle (Sugumaran, 1991, 1998). A mutant lacking phenol oxidase activity is lethal (Asada et al., 1999). Phenol oxidase in insect hemolymph is present as a precursor, prophenol oxidase (Brunet, 1980). In D. melanogaster , two molecular species of prophenol oxidase, A1 and A3, are present, and are activated by a native activator, AMM-1, or by several organic compounds including 2-propanol (Asada et al., 1993b; Chosa et al., 1997; Fujimotoet al., 1993). The activation reaction has been studied using homogeneous prophenol oxidase including the two isoforms or highly purified fraction. Using the null-activity mutants, MoxGM95 andDox-3KD95, our examination of each phenol oxidase activity during Drosophiladevelopment suggests that phenol oxidase has notable effects on the sclerotization of cuticle and hence for its formation.

Thermolabile variant, PHOX-S, of prophenol oxidase in Drosophila melanogaster.

The PhoxS strain of Drosophila melanogaster is an electrophoretically slow variant found in a wild population at Victoria, Australia. Prophenol oxidase isoform A1 from PHOX-S was purified and characterized biochemically and genetically. The purified fraction of A1 from PHOX-S showed a homodimer with a molecular weight of the subunit of approximately 77 kDa. The PhoxS strain was temperature sensitive in vivo in culture, and the purified protein was thermolabile in vitro. By the deletion mapping method, the PhoxS locus was cytologically estimated to be at the location 55-A on the right arm of the second chromosome and 79.6 genetically. These data show that PHOX-S is an electrophoretic variant of MOX and that PHOX-S is the first thermolabile protein found in invertebrate prophenol oxidase.